The klotho gene is known to play a role in suppressing several different aging phenotypes, based on studies in the klotho mutant mouse. Mice homozygous for defects in the klotho gene exhibit a syndrome that closely resembles human aging, including atherosclerosis, osteoporosis, emphysema, infertility and skin atrophy. We have looked for sequence variation in the klotho gene in a paenl of well-characterized mouse strains from the Jackson Laboratory. Each exon of the klotho gene was sequenced in each of these 20 strains. This work is now submitted to the journal Genomics. Specifically, we have found: 1. No variation was found in any of the 16 laboratory derived inbred stains in the panel. 2.Among the 4 wild-derived inbred strains, 45 variants were found, including 43 single nucleotide substitutions, one deletion and one insertion. Of the nucleotide substitutions, six resulted in amino acid substitutions. 3.Real-time RT-PCR analysis of klotho gene expression in the wild-derived strains has shown a higher level of gene expression in SPRET/Ei than in the other wild-derived or laboratory-derived strains. 4. The ratio of membrane form to secreted form of klotho mRNA is higher in the three wild-derived strains with amino acid substitutions that in the control strains. Our finding that the klotho mRNA in the wild-derived species SPRET/Ei is expressed at approximately twice the level of laboratory derived strains and has four amino acid changes is intriguing in light of several phenotypic differences between SPRET/Ei and laboratory derived strains that may be related to klotho expression. These phenotypic differences include a long life-span, exceptional hearing, low total cholesterol and high HDL levels. Interestingly, MOLF/Ei, which exhibited the lowest klotho expression levels of the wild-derived strains, had the highest total cholesterol levels and lowest percent HDL of the 43 strains tested in the mouse phenome project. We hypothesize that klotho mRNA alterations and increased expression levels in SPRET/Ei may provide protection against age-related diseases such as hearing loss and coronary artery disease. It is also possible that specific klotho variants in mice may be associated with increased longevity, as has been seen in humans. Our IRB-approved protocol to look for functional variants of the Klotho gene among participants in the Baltimore Longitudinal Study on Aging was expanded to include the InChianti population as well. We have genotyped the entire BLSA and InChianti populations for the KL-VS allele. Data analysis is underway to search for correlations between specific allelic variants and phenotypes examined in the BLSA and InChianti studies. Over the past year, we have expanded the analysis to include polymorphisms in the LMNA gene in both the BLSA and InCHIANTI populations. We have just completed the analysis of the relationship between the Klotho VS polymorphism in the Baltimore Longitudinal Study of Aing. We found that the polymorpism is associated with insulin resistance (abnormal 2h glucose tollerance test) in men but not in women. Interestingly, both in men and in women, the VS+ polymorphism was not associated with fasting insulin. This is consistent with previous studies that were unable to finds a relationship between Klotho polymorphism and fasting glucose or diabetes diagnosed through abnormal fasting glucose in different representative populations. The data on the Klotho polymorphism in the InCHIANTI study have not been analyzed. The manuscript with the preliminary results was submitted to Nature, but rejected because other studies had found no association between Klotho polymorphysms and glucose metabolism. Thus, we are conducting further analyses aimed at demonstrating that while Klotho affect glucose metabolism during an OGTT test, perhaps by modulating insulin resistance, it does not affect glucose metabolism at reast and this explain the negative results reported by other studies.